The transparency and normal shape of the ocular lens are dependent upon the precise hexagonal geometry, ordered packing, deformability, and membrane organization of highly elongated fiber cells. The broad goals of this research project are to identify and characterize the membrane cytoskeletal scaffolding proteins regulating lens fiber cell shape, cell-cell interactions, membrane stability and organization, and to determine how dysregulation of this scaffolding activity impacts lens transparency and function in animal models. This proposal will investigate the function(s) of the PDZ domain protein-Periaxin, a component of the major cortical adhesive complex-EPPD (ezrin-periaxin-periplakin-desmoyokin) in lens fiber cells. Recent work on characterization of the EPPD complex in the mouse lens led to our discovery that lens fibers exhibit preferential expression of periaxin (PRX), a protein originally thought to be expressed only in myelinating Schwann cells, where it mediates membrane stabilization. PRX expression is robustly induced during fiber cell differentiation, and the protein clusters discretely at the tri-cellular junctions in mature lens fibers. Interestingly, co-immunoprecipitates of PRX from lens fibers contain ankyrin B, spectrin, filensin, desmoyokin, NrCAM and aquaporin-0, suggesting a role for PRX in fiber cell membrane network connectivity, adhesive interactions, and membrane subdomain organization. Significantly, preliminary data from PRX null mice revealed disruptions of fiber cell shape, packing, and membrane integrity but no effects on lens development and growth, arguing a vital role for PRX in maintenance of lens phenotype and function. These significant and novel observations prompted us to hypothesize that the scaffolding interactions of PRX play a key role in stabilization of lens fiber cell tri-cellular junctions, hexagonal geometries, and membrane integrity which are crucial for maintenance of lens transparency and function. For a mechanistic understanding of the role of PRX in mediating the above mentioned functions, three specific aims will be investigated in this application: 1. Analysis of the distribution of different PRX isoforms with unique N-terminal sequences in mouse lens fibers during maturation and compaction, using high resolution confocal 3D imaging;2. Characterization of PRX scaffolding activity in stabilization of fiber cell tri-cellular junctions and membrane organization via identification of PRX interacting proteins using immunoprecipitation, mass spectrometry and yeast two-hybrid analysis;3. Determination of how absence of PRX affects lens optical quality, fiber cell shape, packing and membrane integrity using PRX null and conditional knockout mouse lenses. The completion of these studies is expected to provide novel insights into the broad significance of cortical cytoskeletal scaffolding activity and the specific role of periaxin in maintaining lens fiber cell shape, packing, deformability, cell-cell junctions, and membrane integrity in both normal and cataractous lenses. PUBLIC HEALTH RELEVANCE: Transparency and normal shape are critical for ocular lens function, and rely on the precise packing, deformability, and membrane integrity of hexagonal fiber cells. This application will examine the importance of the cytoskeletal scaffolding activity of periaxin in maintaining lens fiber cell shape, arrangement, and membrane organization using mouse lenses that lack this protein. This project is expected to provide significant insights into the cytoskeletal scaffolding mechanisms regulating lens fiber cell shape, adhesive interactions, arrangement, and membrane organization, and how they may impact optical clarity and function of normal and cataractous lenses.